Kunimaro Furuta

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice.

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

Frequency of, and factors associated with, hepatitis B virus reactivation in hepatitis C patients treated with all-oral direct-acting antivirals: Analysis of a Japanese prospective cohort

Several case reports have shown that hepatitis B virus (HBV) reactivation developed in hepatitis C patients with a current or previous HBV infection during direct‐acting antiviral (DAA) treatment, which led to severe hepatitis or death in some cases. However, its precise frequency and risk factors are not entirely clear. We analyzed a prospective cohort.

Secreted frizzled-related protein 5 (Sfrp5) decreases hepatic stellate cell activation and liver fibrosis

Obesity‐related adipocytokine dysregulation is known to accelerate liver fibrosis progression. Recently, a natural Wnt5a inhibitor, secreted frizzled‐related protein 5 (Sfrp5), was identified as a novel adipocytokine that has reduced expression in obese adipose tissue in both rodents and human. In addition, hepatic gene expression of Wnt5a and its receptor frizzled 2 (Fz2) is elevated during fibrosis progression. Therefore, Sfrp5 could have biological significance in liver fibrosis.

Adiponectin negatively correlates with alcoholic and non‐alcoholic liver dysfunction: Health check‐up study of Japanese men

Aim:  Central obesity, insulin resistance and alcohol consumption are thought to be major risk factors for fatty liver formation. Adiponectin (APN) prevents fatty liver formation, and its serum levels are lower in subjects with central obesity and/or insulin resistance. The aim of this study was to explore the association among serum APN levels, central obesity, insulin resistance and liver dysfunction with or without fatty liver classified by alcohol consumption in healthy subjects.

Pitavastatin ameliorated the progression of steatohepatitis in ovariectomized mice fed a high fat and high cholesterol diet

Many studies indicate an accelerated progression of non‐alcoholic steatohepatitis (NASH) in postmenopausal women. Very recently, we reported that estrogen deficiency enhanced the progression of steatohepatitis in mice fed a high fat and high cholesterol (HFHC) diet. Hypercholesterolemia is often observed in postmenopausal women, and recent studies indicate it to be an important risk factor for the progression of NASH. Statins can slow NASH progression in the estrogen‐deficient state but the precise mechanisms of their effects are still unclear.

Conditional knockout of heparin-binding epidermal growth factor-like growth factor in the liver accelerates carbon tetrachloride-induced liver injury in mice

Aim:  We previously demonstrated that heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is induced in response to several liver injuries. Because the HB‐EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver‐specific HB‐EGF conditional knockout mice using the interferon‐inducible Mx‐1 promoter driven cre recombinase transgene and investigated its role during acute liver injury.

Gab1 adaptor protein acts as a gatekeeper to balance hepatocyte death and proliferation during acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose is the leading cause of drug‐induced acute liver failure. In APAP‐induced acute liver failure, hepatocyte death and subsequent liver regeneration determines the prognosis of patients, making it necessary to identify suitable therapeutic targets based on detailed molecular mechanisms. Grb2‐associated binder 1 (Gab1) adaptor protein plays a crucial role in transmitting signals from growth factor and cytokine receptors to downstream effectors. In this study, we hypothesized that Gab1 is involved in APAP‐induced acute liver failure. Hepatocyte‐specific Gab1 conditional knockout (Gab1CKO) and control mice were treated with 250 mg/kg of APAP. After APAP treatment, Gab1CKO mice had significantly higher mortality and elevated serum alanine aminotransferase levels compared to control mice. Gab1CKO mice had increased hepatocyte death and increased serum levels of high mobility group box 1, a marker of hepatocyte necrosis. In addition, Gab1CKO mice had reduced hepatocyte proliferation. The enhanced hepatotoxicity in Gab1CKO mice was associated with increased activation of stress‐related c‐Jun N‐terminal kinase (JNK) and reduced activation of extracellular signal‐regulated kinase and AKT. Furthermore, Gab1CKO mice showed enhanced mitochondrial translocation of JNK accompanied by an increase in the release of mitochondrial enzymes into the cytosol, which is indicative of increased mitochondrial dysfunction and subsequent nuclear DNA fragmentation. Finally, in vitro experiments showed that Gab1‐deficient hepatocytes were more susceptible to APAP‐induced mitochondrial dysfunction and cell death, suggesting that hepatocyte Gab1 is a direct target of APAP‐induced hepatotoxicity. Conclusion: Our current data demonstrate that hepatocyte Gab1 plays a critical role in controlling the balance between hepatocyte death and compensatory hepatocyte proliferation during APAP‐induced liver injury. (Hepatology 2016;63:1340–1355)

Loss of Gab1 adaptor protein in hepatocytes aggravates experimental liver fibrosis in mice

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 (Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

Nonstructural protein 5A/P32 deletion after failure of ledipasvir/sofosbuvir in hepatitis C virus genotype 1b infection

Nonstructural protein 5A (NS5A) inhibitors play an essential role in the combination treatment of direct-acting antivirals (DAAs) against chronic hepatitis C (CHC). Daclatasvir, a prototype of this class, combined with asunaprevir, has been widely used since 2014 in Asia, where genotype 1b infection is prevalent. Well-known resistance-associated substitutions (RASs) for this treatment include NS5A-L31M/V and Y93H. In addition, recent studies have reported the emergence of a deletion mutant at NS5A-P32 in a subset of patients at the time of virological failure (VF) with this treatment. This mutant is attracting much attention because replicon studies indicate that, compared with NS5A-Y93H, it confers extremely higher resistance to NS5A inhibitors. Moreover, retreatment with DAAs in patients carrying this mutant after daclatasvir/asunaprevir treatment has been reported to be ineffective. Here, we report on a case of NS5A-P32 deletion (P32del) after ledipasvir/sofosbuvir treatment. We treated 1,031 Japanese patients with genotype 1 CHC with ledipasvir/sofosbuvir, and 12 cases exhibited VF. Among these cases, sera from 10 patients available were analyzed for NS5A RAS by deep sequencing, as described (Table 1). All patients had genotype 1b infection and, except for case no. 1, exhibited the NS5A Y93H substitution at baseline or after VF. Case no. 1 showed P32del at the time of VF; the P32del was not detected at baseline (0.1% deep sequencing cutoff), but appeared 4 weeks after completion of 12 weeks of treatment and was continuously detected at a high frequency for 52 weeks (Fig. 1A). After the appearance of the P32del, nucleotide sequences of NS5A domain-I in a major clone were completely consistent at any point. In addition, known RASs to sofosbuvir, including S282T, were not detected by deep sequencing during the entire course. The patient was a 75-year-old treatment-na€ıve man with compensated cirrhosis with a treatment history for hepatocellular carcinoma. We performed phylogenetic tree analysis to clarify the relationship between pretreatment virus and posttreatment virus (Fig. 1B). This analysis indicated that the most frequent wild-type virus at baseline (A1 in Fig. 1B) acquired a deletion of three bases at position P32, becoming virus B18 posttreatment; one more sense mutation resulted in the most frequent virus, B1, posttreatment, which served as the starting point for the subsequent expansion of diversity. Both clinical trials and real-world data have shown that the viral clearance rate of ledipasvir/sofosbuvir is

Increased expression of Forkhead box M1 transcription factor is associated with clinicopathological features and confers a poor prognosis in human hepatocellular carcinoma

Forkhead Box M1 (FoxM1) is a proliferation‐specific transcription factor. In this study, we aimed to elucidate the clinicopathological and prognostic values of FoxM1 expression in human hepatocellular carcinoma (HCC) and correlate FoxM1 expression with various etiologies of liver diseases. We also investigated its therapeutic value in HCC.